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United States Patent | 5,525,482 |
States ,   et al. | * June 11, 1996 |
A method of testing the cytotoxicity and mutagenicity of a chemical includes the steps of exposing test cells to the chemical in vitro, intracellularly metabolizing the chemical into a mutagenic or cytotoxic metabolite and then detecting gene/protein/cell damage in the test cells as an indication of the mutagenicity/cytotoxicity of the chemical. A cell line is provided for testing cytotoxicity and mutagenicity of the chemicals, the cell line consisting essentially of fibroblasts normally having no detectable cytochrome P450 mixed function oxidase enzyme activity. The fibroblasts are transformed with chimeric gene constructs containing cytochrome P450 coding sequences and have intracellular cytochrome P450 oxidative metabolizing activity.
Inventors: | States; J. Christopher (Royal Oak, MI); Hines; Ronald N. (West Bloomfield, MI); Novak; Raymond F. (West Bloomfield, MI) |
Assignee: | Wayne State University (Detroit, MI) |
[*] Notice: | The portion of the term of this patent subsequent to January 19, 2010 has been disclaimed. |
Appl. No.: | 339658 |
Filed: | November 15, 1994 |
Current U.S. Class: | 435/32; 435/29; 435/455 |
Intern'l Class: | C12Q 001/18; C12N 005/10; C12N 015/09 |
Field of Search: | 435/7.21,7.23,29,172.3,240.2,32 536/23.5 |
4696898 | Sep., 1987 | Fitts et al. | 435/69. |
5164313 | Nov., 1992 | Gelboin et al. | 435/189. |
5180666 | Jan., 1993 | States et al. | 435/29. |
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__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 2 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6387 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: complement (2807..3631) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: complement (2125..2251) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: complement (1948..2037) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: complement (1733..1856) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: complement (1501..1587) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: complement (237..1308) (ix) FEATURE: (A) NAME/KEY: promoter (B) LOCATION: complement (3638..3967) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 4586..5446 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCA60 CGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCT120 CACTCATTAGG CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAAT180 TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGACAA240 ACATTAAAAATAGACATTTTATTACAAGAGTGTAGAGAAGGGAGACCAATAGAAG GTAAT300 TGAAATACCCCCCCCTCACTCCAGCCCTAGTCCTGGTGCCTGGATATGTGCACTCCCTGT360 GCGCTCTGATCCCCGCAGACACAAGTCCCCAGCCCCTCCAGGACAGCAATAAGGGTCTTA420 CAAGGCCAGAAGGCAGCCCTGTTTGTTCCT GCCTGCAGGAAGGGCAGAGGAATGTGATGT480 TCCCAGGAACTGTGTCCTAGACCCATAGGGTCAGATTGCTCAGCCTAGTTCAAGCAGTGA540 GACTACCTCTGTGCCAGTATCCTGGGCTGTCTCTTCCCTTCACTCTTGGCAGCTCCCAAT600 TGTC AAAGATTGGACAGGGTCCTGGTTTGGCTAGTTCTAACTTGCTGAAGCCAGTCAGCA660 CCCTCACAGAGCCAGCTAGGTACTGGGCCCAGGGGCTTCCAGAGAGTTCTTCAGAGCTTC720 TCAGAGGCCTAAGGACCTCCTAACCCTAGCAGGCCTCCTGGCTCAAGC ACAACTTGGGAA780 GGCTCCATCAGCATCTATGTGGCCCTGTTTTACCTGTTGTCTCTGGAGGGTGTGCAGAGG840 CAAGTCCAGGGTAGGGGCAGGCAGGATCCCTTAGGCTTGCCCACAGCCCAGATAGCAAAA900 CTGCAGCCAGATCAGTGTCTAT GAGTTTCAGGCTGAATCTTAGACCACATAGGCCAGCCT960 GCTGGTCTGGCTGCCCAACCAGACCAGGTAGACAGAGTCTAGGCCTCAGGGCTCTCAAGC1020 ACCTAAGAGCGCAGCTGCATTTGGAAGTGCTCACAGCAGGCATGCTTCATGGTTAGCCCA1080 TAGATGGGGGTCATGTCCACCTTCACGCCCAGTGGCACGCTGAATTCCACCCGTTGCAGC1140 AGGATAGCCAGGAAGAGAAAGACCTCCCAGCGGGCAATGGTCTCACCGATACACTTCCGC1200 TTGCCCATGCCAAAGATAATCACCTTCTCACTTAACACCT TGTCGATAGCACCATCAGGG1260 GTGAGAAACCGTTCAGGTAGGAACTCAGATGGGTTGACCCATAGCTTCCTGTAACCAGAG1320 GGAGACAGCTGAAGTGGCAGTTCAGGGCTCAGAAGTGTCAAGTGAGTGGAGCTCCAGCCC1380 CAAAGGATAGAGGAC AGGCAAGCAGCCCATGGACAGGAGGATCAATGCAATGATTGTATT1440 AATCATATATAAGAGCTTAAGAGGGTGGACCCAGCCTTTCCTCTGCATCTCTGAACTTAC1500 TGGTCATGGTTGATCTGCCACTGGTTTACAAAGACACAACGCCCCTTGGGGATGTAAAA G1560 CCTTTCAAACTTGTGTCTCTTGTTGTGCTAGGGAGAAAGGAAGCTCAGTCAGGCTCAGGG1620 CAACAGGCAAATCTCCCTGTCTCCCATGCCGTGTCCCTCCCACTAACCCTAATCAGGTAT1680 GTGGTCCGGAGTAAGATCAGTAACAGACAGCAG TGGCTCCATGGGGCCTTACCTGTGGGG1740 GATGGTGAAGGGGACGAAGGAAGAGTGTCGGAAGGTCTCCAGGATGAAGGCCTCCATATA1800 GGGCAGATGGGATCTGTCAGAGAGCCGGGGCCGCCGTGACCTGCCAATCACTGTGTCTGC1860 AGAACACA GGGACAAGATGGATGCAGGGGCTGCCTAGACCTGGCCAGACCCCTGGCACTG1920 ACCCCTTTGAAGGGAGCCACTACCTACCTAGCTCCTCTTGGATCTTTCTCTGTACCCTGG1980 GGTTCATCACCAAATACATGAGGCTCCAGGAGATAGCAGTTGTGACTGTGT CAAACCCTG2040 GACAGGGTAGAACAGAAGAAGTTAGGCAGGCAGCAGCAGGTCAGGGCACTTGAGCACAGG2100 AAGGACACAATGGGGTAACCATACCAGCTCCAAAGAGGTCCAAGACGATGTTAATGATCT2160 TCTCATCTGACAGCTGGACATTGGCG TTCTCATCCAGCTGCTTCTCCTGACAGTGCTCAA2220 TCAGGCTGTCTGTGATGTCCCGGATGTGGCCCTTAGGTAGGGAAAGTCCACAGGTGAGCA2280 AGATCTCAAACCCAGAGCTACCTCTCCATCCAGGTCTGGTCCTTCACTATTCCTAGCACA2340 TTTGTTCTGGAGGTGATGCCCCCTGAGGCTGTTGTCCCAGCTTCTCTCTGCCTCTGCAAG2400 GCTCTCTCCTACTACCTTAGAATGCTGCTAGCCCCAACTCATGGGACATTTGACACACAG2460 CGTCTTGTACTGTTATCATCTGGATGTGCTCTTTATCTTCTTTC TATTATGAGGCCAGGA2520 GATGAACTCTTTAACTCTTCCTGGCTCCCTCAGCAACTGCCCCAGGGTCCTGCATGTAAT2580 GACTCTTCAGTGGCTATTGCTGTCTGTGGAAGCATGGAAGGGTTAGTCAAGATAAAGTTC2640 TATTTCCCTGCCAAGGAAG AAGACTATTCCACAACTGGCTTCAAGATCCCAGGTTGAAGC2700 CTTCCTGAGAACTTGCCAAGCCCCATGCAGTTCCTCTTACCTTTGACCTCCCAGGCCCTG2760 ATGCCATCTGCTTCCCACCACCCACCTGCCTTTCCCCAGACTGTACCTTCTCAAAGGTTT 2820 TGTAGTGCTCCTTGACCATCTTCTGCATGAAGCTGTAGAACTTCTCATTCAGGTCCTTGA2880 AGGCATTCAGGGAAGGGTTGGGTAGGTAGCGAAGAATAGGGATGAAGTCAGCTGGGTTTC2940 CAGAGCCAACCACCTCCCCGAAATTATTATTCAGGTT GACTAGGCTAAGCAGTTCTTGGT3000 GGTTGTGGTCATAGCGCCGGCCAAAGCAAATGGCACAGATGACATTGGTCACTGATACCA3060 CCACATACCTGTAGGGGTTAAAGTGCCCAGGCCCTGCCATCAGCTCCTGCAACGTGCTTA3120 TCAGGACCTCA GCCTCCTTGCTCACATGCTCTTCCAGGTAGCAGGAGGTTGAGGAGGCTG3180 GGTCAGAGGCAATGGAGAAACTTTTCAGGCCATTCTGGGCCAGGCGCCGGCGGGCAGCCC3240 ACACTGGTCCAGAGTCTGGGCTGAAGGACATGCTCTGACCATTACTGATGAGGGT GAAGG3300 TGTAGAGGTCGGGCCGGCCCTTGAAATCATCGCCCTGCCGCACCAGGGCCTGCCGGATGG3360 TGTCCAGGCCGCTCAGCACCACCACGGGTGTGGAGCCAATTCGGATCTGCAGCACGTCCC3420 CATACTGCTGGCTCATCCTTGACAGTGCCA GGTGCGGGTTCTTTCCCAGGGTCAGCATGT3480 GCCCAATCAGAGGCCAGCCCCATGGCCCTGGTGGATTCTTCAGGCCTTTGGGGACCTGAG3540 GTCTTGAGGCCCTGATTACCCAGAATACCAGACAGAAGATGACAGAGGCCAGAAGAAACT3600 CCGT GGCCGACATGGAGATTGGGAAAAGCATGATCTGGTGAAGCTGGAGCTACGGAGTAA3660 GTGAGGAGAAGGTACTCAGGACGTTGAAGTCGTGGTGACGCTTAGAGGACAGCCTGCCCT3720 CTTTATAGTCGTTGGACGAGTCCGGGCGCAAAGGGTTTGCACCCAGCA GGCGGTTGCTCC3780 AGCCCACGCATAGTCACGCGCCCCGCGTCCTGGCAGAGCCGAGCGCACTTTTCGGGCGGA3840 GTGCAGAGCTCCCTGGAGCGCCAGTGTGCACAGCGGGCACGCCCCTTTGGCACGACTGGC3900 TCGGATAGGACGACCCATGTGA CGTGTGGAACATCATGTCCCTATAGTGCTTTCCCCGAT3960 ATTACGGGGGCGAGCTCGAATTAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTG4020 GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTG4080 GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG4140 CGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAT4200 ATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG TTAAGCCAGCCCCGACACCC4260 GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACA4320 AGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACG4380 CGCGAGACGAAAGGG CCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAAT4440 GGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTT4500 ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGC T4560 TCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC4612 MetSerIleGlnHisPheArgValAla 15 CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCA4660 LeuIleProPhePheAlaAlaPheCysLeuProValPheAlaHisPro 101520 25 GAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA4708 GluThrLeuValLysValLysAspAlaGluAspGlnLeuGlyAlaArg 3035 40 GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGT4756 ValGlyTyrIleGluLeuAspLeuAsnSerGlyLysIleLeuGluSer 4550 55 TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG4804 PheArgProGluGluArgPheProMetMetSerThrPheLysValLeu 6065 70 CTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTC4852 LeuCysGlyAlaValLeuSerArgIleAspAlaGlyGlnGluGlnLeu 758085 GGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA4900 GlyArgArgIleHisTyrSerGlnAsnAspLeuValGluTyrSerPro 9095100 105 GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGC4948 ValThrGluLysHisLeuThrAspGlyMetThrValArgGluLeuCys 110115 120 AGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTG4996 SerAlaAlaIleThrMetSerAspAsnThrAlaAlaAsnLeuLeuLeu 125130 135 ACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATG5044 ThrThrIleGlyGlyProLysGluLeuThrAlaPheLeuHisAsnMet 140145 150 GGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA5092 GlyAspHisValThrArgLeuAspArgTrpGluProGluLeuAsnGlu 155160165 GCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCA5140 AlaIleProAsnAspGluArgAspThrThrMetProValAlaMetAla 170175180 185 ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCC5188 ThrThrLeuArgLysLeuLeuThrGlyGluLeuLeuThrLeuAlaSer 190195 200 CGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCA5236 ArgGlnGlnLeuIleAspTrpMetGluAlaAspLysValAlaGlyPro 205210 215 CTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT5284 LeuLeuArgSerAlaLeuProAlaGlyTrpPheIleAlaAspLysSer 220225 230 GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCA5332 GlyAlaGlyGluArgGlySerArgGlyIleIleAlaAlaLeuGlyPro 235240245 GATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAG5380 AspGlyLysProSerArgIleValValIleTyrThrThrGlySerGln 250255260 265 GCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCA5428 AlaThrMetAspGluArgAsnArgGlnIleAlaGluIleGlyAlaSer 270275 280 CTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG5483 LeuIleLysHisTrp 285 ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA 5543 TGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGA5603 TCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAA5663 AACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA AGAGCTACCAACTCTTTTTCCGA5723 AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGT5783 TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT5843 TACCAGTGGCT GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGAT5903 AGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT5963 TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG CGCCA6023 CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG6083 AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTC6143 GCCACCTCTGACTTGAGCGTCGATTTTTGT GATGCTCGTCAGGGGGGCGGAGCCTATGGA6203 AAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACA6263 TGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAG6323 CTGA TACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG6383 AAGA6387 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 286 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetSerIleGlnHisPheArgValAlaLeuIleProPhePheAlaAla 151015 PheCys LeuProValPheAlaHisProGluThrLeuValLysValLys 202530 AspAlaGluAspGlnLeuGlyAlaArgValGlyTyrIleGluLeuAsp 35 4045 LeuAsnSerGlyLysIleLeuGluSerPheArgProGluGluArgPhe 505560 ProMetMetSerThrPheLysValLeuLeuCys GlyAlaValLeuSer 65707580 ArgIleAspAlaGlyGlnGluGlnLeuGlyArgArgIleHisTyrSer 8590 95 GlnAsnAspLeuValGluTyrSerProValThrGluLysHisLeuThr 100105110 AspGlyMetThrValArgGluLeuCysSerAlaAlaIleThrM etSer 115120125 AspAsnThrAlaAlaAsnLeuLeuLeuThrThrIleGlyGlyProLys 130135140 GluLeuThrAlaPhe LeuHisAsnMetGlyAspHisValThrArgLeu 145150155160 AspArgTrpGluProGluLeuAsnGluAlaIleProAsnAspGluArg 165 170175 AspThrThrMetProValAlaMetAlaThrThrLeuArgLysLeuLeu 180185190 ThrGlyGluLeuLeuThrLeuAla SerArgGlnGlnLeuIleAspTrp 195200205 MetGluAlaAspLysValAlaGlyProLeuLeuArgSerAlaLeuPro 2102152 20 AlaGlyTrpPheIleAlaAspLysSerGlyAlaGlyGluArgGlySer 225230235240 ArgGlyIleIleAlaAlaLeuGlyProAspGlyLysProSerArgI le 245250255 ValValIleTyrThrThrGlySerGlnAlaThrMetAspGluArgAsn 260265270 ArgGln IleAlaGluIleGlyAlaSerLeuIleLysHisTrp 275280285 __________________________________________________________________________